Lignin is a complex phenolic polymer that fortifies plant cell walls and is essential for plant growth and development. The presence of lignin in biomass, however, acts as a major impediment to industrial processing. Research efforts have therefore focused on altering the natural lignification processes to produce plants with cell walls that process more readily to liberate carbohydrates with minimal input (Li et al. 2008, Chen et al. 2007, Vanholme et al. 2008, Ralph et al. 2004, Boerjan et al. 2003).
The biosynthetic steps to produce the monomers employed in the synthesis of lignin have been elucidated (Li et al. 2008, Ralph et al. 2004, Boerjan et al. 2003). New genes relevant to lignin production continue to be discovered (Vanholme et al. 2008, Withers et al. 2012), and several transcription factors integral to controlling the lignin biosynthetic network have been identified (Li et al. 2012). Perturbations of these and other processes can lead to the synthesis of lignins that incorporate alternative monomers. These monomers are usually derived from products of incomplete monolignol biosynthesis. These discoveries spawned the idea that lignins could be designed to encompass significant structural alterations that would engender unique properties (Ralph et al. 2004, Vanholme et al. 2012, Ralph 2010).
Studies of natural plant tissues, along with those from mutants and transgenics with misregulated monolignol biosynthetic genes, have led to some remarkable discoveries, including plants that produce homopolymers from a range of “traditional” monomers (e.g., p-coumaryl and sinapyl alcohols (Stewart et al. 2009, Bonawitz et al. 2014)) as well as “non-traditional” monomers (e.g., caffeyl and 5-hydroxyconiferyl alcohols, and the hydroxycinnamaldehydes (Chen et al. 2012, Vanholme et al. 2010, Weng et al. 2010, Zhao et al. 2013). These observations clearly illustrate the pliability of the lignification process (Ralph et al. 2004, Ralph 2010, Ralph et al. 2008). The formal “design” of an improved polymer using unconventional monomers therefore seems to be a feasible path to tailor plants with superior processing properties for both paper and biofuels production (Vanholme et al. 2012, Ralph 2010, Wilkerson et al. 2014, Grabber et al. 2008).
Redesigning lignin to be more amenable to chemical depolymerization can lower the energy required for industrial processing. In this vein, lignin has been engineered to contain readily cleavable ester bonds in the form of monolignol ester conjugates in the polymer backbone, improving its degradability. See U.S. Pat. No. 8,569,465, US 2011/0003978, US 2013/0203973, US 2013/0219547, US 2015/0020234, WO 2012/012698, WO 2013/052660, and WO 2013/090814. Poplar trees, for example, have been engineered to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates (Wilkerson et al. 2014). Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrated that these trees produce the monolignol ferulate conjugates, export them to the cell wall, and utilize them during lignification.
Tailoring plants to employ monolignol ester conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction. Methods for directly detecting and determining levels of such monolignol ester conjugates incorporated into plant lignin, however, are not known in the art and are therefore needed.